目前支持人类胚胎干细胞研究在Teitell实验室由加州再生医学研究所（CIRM）种子批RS1 - 00313。我们感谢卡拉克勒博士（加州大学洛杉矶分校）和克勒实验室成员在这些问题和进一步的研究提供意见和见解。

高分辨率清除母语凝胶Electophoresis线粒体复合物（hrCNE）活性测定 - 凝胶单层ˠ照射或丝裂霉素C处理过的小鼠胚胎成纤维细胞（MEFs）然后切换MEF空调介质中，使用标准的条件下5天的基质胶上的增长，对维持人类胚胎干细胞（胚胎干细胞）。为了去除污染MEFs，人类胚胎干细胞被重新镀上基质胶和MEF空调额外3天的中期增长。 Oct - 4和SSEA - 4的多能性标志物免疫荧光显微镜是用来确认的人类胚胎干细胞分化的缺乏。 <ol><li>细胞与温暖的1X PBS，pH值7.4，胰蛋白酶（1毫升1X胰蛋白酶在6孔板，每孔）5分钟洗净。 </li><li>添加新鲜的胰蛋白酶抑制剂等体积（1mg/ml的1X PBS，pH值7.4）。在200克收获细胞和离心5分钟，室温。 </li><li>丢弃在0.8上清液和悬浮颗粒细胞 - 1.7毫升冰冷的线粒体隔离缓冲区（83MM蔗糖; 6.6mM咪唑/盐酸，pH值7.0）（根据细胞沉淀的大小）新鲜配制的1蛋白酶抑制剂。在冰上孵育10分钟。 </li><li> Dounce细胞与均质机（〜40 - 50杆）。 dounced细胞台盼蓝染色等份，以同质化的使用光学显微镜的完整性检查。 </li><li>在20000克离心10分钟，4 ° C至获得原油线粒体还包含细胞核和细胞碎片较大的颗粒。倒出并丢弃上清液，并权衡沉淀原油线粒体颗粒。颗粒可以保存于-80 ° C以下液氮的瞬间凝固，如果这是一个停止点。 注：可以用于分离和收集一个纯粹的线粒体膜分数差速离心或其他基于梯度程序。见文献。有关详细信息，4。这种替代的方法将产生每毫克线粒体蛋白复杂的活动，而不是每毫克体重细胞或细胞数量的线粒体复杂的活动。线粒体蛋白的浓度，将需要净化，使这个比较后确定。此外，这种方法可能需要不同的隔离缓冲。溶解纯线粒体膜分数，请注意以下步骤6。 </li><li>冰冰冷的增溶缓冲液（氯化钠50MM，50MM咪唑/盐酸pH值7.0，为1mM EDTA，2MM 6 - 氨基己酸）涡旋溶解原油线粒体颗粒。 6 - 氨基己酸，可以被替换为1个蛋白酶抑制剂。使用颗粒重量的20毫克每增溶缓冲液35μl。加入20μL的10％W / V每20毫克的颗粒重量和组合由轻弹管毛地黄皂苷。在冰上孵育5-10分钟。十二烷基- BD -麦芽糖或Triton X - 100可替代毛地黄皂苷溶解的蛋白质。洗涤剂和数量的选择可能会影响supercomplexes线粒体复合物多聚体形式的形成。欲了解更多信息，请参阅参考资料1。 注：所需数量的洗涤剂溶解一个纯粹的线粒体膜分数对应的洗涤剂/蛋白质2 - 6G /克不等的重量比。溶解线粒体膜的最佳比例是2.5克/克为十二烷基- BD -麦芽糖，3G / G为Triton X - 100，和6g /毛地黄皂苷克。 </li><li> 10万克离心15分钟在4 ° C溶解的沉淀收集上清液。 </li><li>样缓冲液加入到最后的5％W / V甘油和0.01％W / V丽春红（使用50％W / V甘油，0.1％W / V丽春红加载缓冲库存解决方案）。 </li><li>装载卷包含原油线粒体颗粒每通道20 - 40毫克凝胶井。使用3.5％的浓缩胶，丙烯酰胺梯度凝胶电泳（4 -13％）。 注：凝胶的制备及电泳条件： 凝胶法制备，使用如下的丙烯酰胺/双丙烯酰胺（AA / bisAA）3组合：丙烯酰胺和双丙烯酰胺1.5克（49.5％T，3％，其中T是AA和bisAA和C总浓度是48克总单体交联剂（bisAA）的百分比）。 胶缓冲液（3）：1.5M 6 - 氨基己酸; 75MM咪唑，pH值7.0 </li></ol><table style="padding-left: 60px;" border="0"><tbody><tr><td>梯度凝胶： </td><td> 4％ </td><td> 13％ </td><td>浓缩胶： </td><td> 3.5％ </td></tr><tr><td> AA / bisAA 3混合</td><td> 2毫升</td><td> 6.5毫升</td><td></td><td> 0.6毫升</td></tr><tr><td>凝胶缓冲3“ </td><td> 8.3毫升</td><td> 8.3毫升</td><td></td><td> 2.7毫升</td></tr><tr><td>甘油</td><td></td><td> 5克</td><td></td><td></td></tr><tr><td> H 2 O </td><td> 14.7毫升</td><td> 5毫升</td><td></td><td> 4.7毫升</td></tr><tr><td>总成交量</td><td> 25毫升</td><td> 25毫升</td><td></td><td> 8毫升</td></tr><tr><td> 10％的APS </td><td> 139μL </td><td> 125μL </td><td></td><td> 67μL </td></tr><tr><td> TEMED </td><td> 14μL </td><td> 12.5μL </td><td></td><td> 6.7μL </td></tr></tbody></table>阳极缓冲：25MM咪唑/盐酸，pH值7.0 阴极缓冲液：50MM，7.5MM咪唑甘氨酸/盐酸，0.02％W / V十二烷基- BD -麦芽糖，0.05％，pH值7.0 W / V脱氧胆酸 在寒冷的房间（4-7℃）倒入和运行凝胶。所使用的初始电压是100V。当样品进入浓缩胶，引发电压与电流限制到500V至15mA（0.15&#39;14“14厘米凝胶）。电泳后3-4小时，当红色的丽春红染料的急剧行的方法凝胶前停止。另外，凝胶可以用恒压运行在70 - 100V过夜。 <ol><li>凝胶活性测定。每次检测的孵化时间取决于蛋白质增溶的效率和材料的数量，每孔装。我，IV和V配合物通常会产生更强的信号比复合物II和III。 </li></ol>复杂的I（CI） 孵育CI缓冲区（5MM的Tris -盐酸，pH值7.4 2.5mg/ml NBT;为0.1mg/ml NADH）的凝胶片在室温下了好几个小时。 CI为缓冲区：每5MM的Tris - HCl，pH值7.4百毫升添加： 0.01克NADH 0.25克NBT 修复凝胶在50％甲醇和10％醋酸为15-45分钟。固定凝胶是保存在10％醋酸。 复合物Ⅱ（CII） 孵育与CII的缓冲区（20MM琥珀酸钠5MM的Tris - HCl pH值7.4; 0.2MM吩嗪methasulfate; 2.5mg/ml NBT）凝胶片几个小时，在室温下。 CII的缓冲区：每5MM的Tris - HCl，pH值7.4100毫升添加： 200μL1M琥珀酸钠 80μl吩嗪methasulfate（250MM的PMS DMSO溶解） 0.25克NBT 修复复杂所述的凝胶综合三（CIII） 孵育CIII缓冲区（50MM磷酸钠，pH值7.2; 0.05％3,3&#39; - 二氨基tetrahydrochloride（DAB））的凝胶片在室温下了好几个小时。 CIII缓冲液：每50毫米的钠磷酸盐，pH值7.2100毫升： 50毫克二氨基tetrahydrochloride 修复复杂所述的凝胶综合四（CIV） 孵育CIV缓冲区（50MM磷酸钠，pH值7.2; 0.05％3,3&#39; - 二氨基tetrahydrochloride（DAB），50μM马心细胞色素c）的凝胶片几个小时，在室温下。 CIV缓冲液：每50毫米的钠磷酸盐，pH值7.2百毫升： 50毫克二氨基tetrahydrochloride 0.1克马心细胞色素c 修复复杂所述的凝胶综合五（简历） 孵育凝胶片CV缓冲区（35MM三基地，270甘氨酸，pH值8.3在25 ° C，14mm的硫酸镁 ，0.2％W / V PB（NO 3）2，8mm的三磷酸腺苷）在室温下了好几个小时。 CV缓冲液：每100毫升的解决方案中添加： <table border="0"><tbody><tr><td>三基</td><td> 0.424克</td></tr><tr><td>甘氨酸</td><td> 2.026克</td></tr><tr><td> 硫酸镁 </td><td> 0.345克</td></tr><tr><td> PB（NO 3）2 </td><td> 0.29克</td></tr></tbody></table>无需调整pH值（应该是8.3） - 不使用酸或碱。过滤和添加<table border="0"><tbody><tr><td>三磷酸腺苷</td><td> 0.441克</td></tr></tbody></table>修复凝胶在50％甲醇30分钟，并转移到水。

<ol>
	<li>Wittig, I., Braun, H. -. P., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nature Protocols. 1, 418-428 (2006).</li><li>Wittig, I., Karas, M., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Resolution+Clear+Native+Electrophoresis+for+In-gel+Functional+Assays+and+Fluorescence+Studies+of+Membrane+Protein+Complexes.++6.">High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes. 6.</a> Molecular &amp; Cellular Proteomics. 6, 1215-1225 (2007).</li><li>Wittig, I., Carrozzo, R., Santorelli, F. M., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assays+in+high-resolution+clear+native+gels+to+quantify+mitochondrial+complexes+in+human+biopsies+and+cell+lines.">Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines.</a> Electrophoresis. 28, 3811-3820 (2007).</li><li>Castle, J. D., Coligan, J. E., Dunn, B. M., Speicher, D. W., Wingfield, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Organelles+from+Mammalian+Cells.">Purification of Organelles from Mammalian Cells.</a> Current Protocols in Protein Science. 4.2, 4.2.1-4.2.57 (2007).</li></ol>

在这段视频中，我们已经描述了从人类胚胎干细胞，他们高分辨率的清晰本地凝胶电泳（hrCNE）分离，以及随后的分析凝胶的催化活性检测线粒体蛋白质复合物的提取。 hrCNE解析线粒体膜蛋白复合物，并在一项决议，与蓝色本地凝胶电泳凝胶活性测定优越。可以采用这种技术不仅量化线粒体呼吸复合物IV也分析，无论是在人类胚胎干细胞和其衍生的后代的任何线粒体膜蛋白复合物的亚基组成。

<table><tbody><tr><td>0.5M EDTA</td><td>Fisher Scientific</td><td>BP120-1</td><td>adjust pH to 8.0 with NaOH </td></tr><tr><td>1M NaCl</td><td>Sigma-Aldrich</td><td>S3014-500MG</td><td></td></tr><tr><td>1M Sodium succinate</td><td>Fisher Scientific</td><td>S413-500</td><td></td></tr><tr><td>0.1M Sodium phosphate buffer pH 7.2</td><td>Fisher Scientific</td><td>BP332-1 and BP329-1</td><td>Mix 68.4 ml of 1M Na2HPO4 and 31.6 ml of 1M NaH2PO4 to prepare 0.1M sodium phosphate buffer with pH 7.2 at 25 C </td></tr><tr><td>1M Tricine, pH 7.0 (adjusted with NaOH)</td><td>EMD Millipore</td><td>9010</td><td>store at 4 C </td></tr><tr><td>1M imidazole/HCl, pH 7.0</td><td>Sigma-Aldrich</td><td>I-2399</td><td>store at 4 C</td></tr><tr><td>1M Tris-HCl, pH 7.4</td><td>Fisher Scientific</td><td>BP153-1</td><td></td></tr><tr><td>20% (wt/vol) dodecyl-b-D-maltoside (DDM)</td><td>Fluka</td><td>44205-500MG</td><td>Dissolved in water. Store 1 ml aliquots at &amp;#8211;20 C.</td></tr><tr><td>10% (wt/vol) digitonin (&amp;gt;50% purity, used without recrystallization)</td><td>Fluka</td><td>37008-1G</td><td>Dissolved in water. Store 0.1&amp;#8211;1 ml aliquots at &amp;#8211;20 C. Should be warmed up to 95 C before use.</td></tr><tr><td>2M 6-aminohexanoic acid</td><td>Fluka</td><td>07260-100G</td><td>store at 4 C</td></tr><tr><td>10x Ponceau S/glycerol stock solution</td><td>Sigma-Aldrich</td><td>P-3504 and G6279-1L</td><td>0.1% Ponceau S, 50% glycerol, (wt/vol) in water.</td></tr><tr><td>10% w/v sodium deoxycholate</td><td>Fluka</td><td>30970-25G</td><td>Dissolved in water. Must be protected from light. Adjust pH to 7.0 with HCl, filter and store at room temperature.</td></tr><tr><td>250 mM phenazine methasulfate (PMS)</td><td>TCI America</td><td>P0083</td><td>Dissolved in DMSO and aliquoted. Store at &amp;#8211;20 C.</td></tr><tr><td>Nitro-blue tetrazolium (NBT)</td><td>Amresco</td><td>0329-1G</td><td></td></tr><tr><td>3,3&amp;rsquo;-diaminobenzidine tetrahydrochloride (DAB)</td><td>TCI America</td><td>D0078</td><td></td></tr><tr><td>Pb(NO3)2</td><td>Fisher Scientific</td><td>L62-100</td><td></td></tr><tr><td>MgSO4</td><td>Fisher Scientific</td><td>BP213-1</td><td></td></tr><tr><td>Tris base </td><td>Fisher Scientific</td><td>BP152-10</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>G45-212</td><td></td></tr><tr><td>Adenosine 5&amp;rsquo;-triphosphate disodium salt (ATP)</td><td>Sigma-Aldrich</td><td>A2383-5G</td><td>Grade I, minimum 99%</td></tr><tr><td>beta-nicotineamide adenine dinucleotide (NADH) reduced form </td><td>Sigma-Aldrich</td><td>N-8129</td><td></td></tr><tr><td>Cytochrome c From horse heart</td><td>Sigma-Aldrich</td><td>C2506-250MG</td><td></td></tr><tr><td>Sucrose </td><td>Fisher Scientific</td><td>BP220-212</td><td></td></tr><tr><td>100x protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P8340</td><td>Store at &amp;#8211; 20 C.</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>D128-500</td><td></td></tr><tr><td>Trypan blue Stain 0.4%</td><td>GIBCO, by Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>10x Trypsin (0.5%)</td><td>GIBCO, by Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>Trypsin Inhibitor </td><td>GIBCO, by Life Technologies</td><td>17075-029</td><td></td></tr></tbody></table>

probing for mitochondrial complex activity in human embryonic stem cells

线粒体是细胞质的细胞器，有一个细胞代谢和动态平衡的主要作用，细胞信号转导网络调控，和程序性细胞死亡。线粒体产生ATP，调节细胞质的氧化还原状态和Ca2 +的平衡，分解代谢脂肪酸，合成血红素，核苷酸，类固醇激素，氨基酸，并帮助组装蛋白的铁硫簇。线粒体也有产热的重要作用。线粒体基因组的突变引起人类疾病的几种类型。 mtDNA突变的积累与衰老相关，怀疑是在癌症发展的重要作用。由于他们的作用极为重要，在所有类型的细胞，线粒体的功能，还必须干细胞的关键。干细胞活力，增殖和分化能力的认识已经取得了重要进展。但干细胞的功能活动，尤其是他们的能量状态，尚未被详细研究。几乎没有什么是已知的人类胚胎干细胞（胚胎干细胞），和他们的区别的易制毒化学后代线粒体属性。线粒体在人类胚胎干细胞的功能和发展潜力作用来认识和评价的方法之一是直接测定线粒体呼吸复合物的活性。在这里，我们描述了高分辨率的清晰的原生凝胶电泳凝胶作为活动的可视化分析五年的人类胚胎干细胞呼吸链复合物的方法和后续。

Watch this Scientific Journal Video about Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells at JoVE.com

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

在这段视频中，我们将向您可以分析人类胚胎干细胞的线粒体呼吸链复合物如何使用凝胶活性测定。

在人类胚胎干细胞的线粒体复杂的活动初探

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

probing-for-mitochondrial-complex-activity-human-embryonic-stem

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.2K 次观看.  University of California, Los Angeles. 在这段视频中，我们将向您可以分析人类胚胎干细胞的线粒体呼吸链复合物如何使用凝胶活性测定。

视频: Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

用天然电泳，凝胶电泳和电解法分析线粒体电子传递链的超复合物

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

科研

生物化学

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

混合光/蓝色原生电泳在线粒体呼吸链超综合体分离与分析中的作用

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

测量来自母猪后代的先前冷冻心脏组织中的线粒体电子转移复合物:评估运动诱导的线粒体生物能量变化的模型

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

稳定同位素追踪和LC-MS分析以测量DHODH和SDH活性

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

作者聚焦：测量哺乳动物线粒体功能的氧非依赖型检测  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

测量哺乳动物线粒体功能的氧非依赖性测定

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular mechanisms that maintain oocyte quality remain unclear, thus it is difficult to increase the birth rate of women over 35 years old at present. Oocytes contain more mitochondria than any type of cell in the body, and any mitochondrial dysfunction can lead to reduced oocyte quality. In the 1990s, oocyte cytoplasmic transfer resulted in great success in human reproduction but was accompanied by ethical controversies. Autologous mitochondrial transplantation is expected to be a useful technique to increase the quality of oocytes that have decreased due to age. In the present study, we used adipose-derived stem cells from aged mice as a mitochondria donor to increase the quality of oocytes of aged mice. Further development of autologous mitochondrial transfer technology will provide a new and effective treatment for infertility in aged women.

Here, we describe the isolation of mitochondria from mouse adipose-derived mesenchymal stem cells, and then transfer the mitochondria into aged mouse oocytes to improve the quality of the oocytes.

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular ...

发育生物学

用于超复合物分析的小组织样品中的线粒体分离

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

作者聚焦：揭示氧化磷酸化系统动力学和线粒体在健康和疾病中的作用

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析 

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

在人类胚胎干细胞的线粒体复杂的活动初探

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用天然电泳，凝胶电泳和电解法分析线粒体电子传递链的超复合物

混合光/蓝色原生电泳在线粒体呼吸链超综合体分离与分析中的作用

测量来自母猪后代的先前冷冻心脏组织中的线粒体电子转移复合物:评估运动诱导的线粒体生物能量变化的模型

测量哺乳动物线粒体功能的氧非依赖性测定

测量哺乳动物线粒体功能的氧非依赖性测定

测量哺乳动物线粒体功能的氧非依赖性测定

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析